Kit for diagnosing GH deficiency

ABSTRACT

A kit for diagnosis of autosomal dominantly inherited isolated growth hormone (GH) deficiency (IGHD II) in a patient sample comprises means for analyzing a patient sample for the presence or absence of a T at nucleotide position 6191 in exon 4 of the GH-1 gene.

RELATED APPLICATIONS

The present application is a continuation of application Ser. No.10/137,578 filed May 2, 2002, and claims priority under 35 U.S.C. §119of U.S. application Ser. No. 60/288,190 filed May 2, 2001.

BACKGROUND OF THE INVENTION

The majority of cases with isolated growth hormone deficiency (IGHD) areidiopathic (1). Monogenetic recessive inheritance of IGHD was shown tobe caused by complete deletions of the GH-1 (1GHD IA)(2) and, morerecently, by nonsense mutations of the GHRH receptor gene (3). Dominanttransmission (IGHD II) was exclusively found in the presence of GH-1splice site mutations which cause skipping of exon 3 (4,5). Thisin-frame deletion results in the loss of 40 amino acids and a presumablymisfolded de 132-71 GH. The prevalence of such mutations in familieswith IGHD II is high, up to 100% (6). The mechanism of the dominantnegative effect of the mutant protein is only partly understood (7).In-vitro studies suggested cell-specific mechanisms in neuro-endocrinecells which included insufficient storage and secretion of the wild-typeGH in the presence of the del32-71GH (8,9). Seven different splice sitemutations in intron 3 of GH-1 have been reported (4,5,10-13). Because ofthe very compact gene structure of the GH-1, splicing is also affectedby point mutations outside the conserved splicing sites (14). Inaddition, two GH-1 missense mutation (P89L and R183H) were recentlyimplicated in IGHD II (15,16).

SUMMARY OF THE INVENTION

The present invention provides novel markers for diagnosing IGHD II.These markers provide early and accurate diagnosis of affected children.The present inventors have found that not only splice site mutationscausing skipping of exon 3 of GH-1 but also GH-1 missense mutationsresult in a mutant GH with a dominant negative effect. Thus it is veryimportant to also investigate children with suspected GH-deficiency formissense mutations.

In a first aspect, the invention relates to a method for diagnosis ofautosomal dominantly inherited isolated GH deficiency (IGHD II) in apatient sample, comprising in vitro analysing the presence or absence ofthe novel missense mutation G6191 to T in exon 4 of GH-1 which changesvaline 110 to phenylalanine. Preferably, the method also comprisesanalysing the presence or absence of GH-1 splice site mutations causingskipping of exon 3 of GH-1.

In one preferred embodiment the splice site mutation is the novel +2T toC transition of the second base of the intron 3 donor splice site.

Thus, the present invention provides two novel disease markers for IGHDII.

The method of the invention preferably comprises amplification of theGH-1 gene of the patient or fragments derived from the GH-1 gene.Preferably, the intron 3 and/or exon 2-5 are amplified.

In a preferred embodiment, the method comprises PCR amplification of theGH-1 gene of the patient and nested PCR of overlapping constituentfragments of the GH-1 gene of the patient.

The amplified fragments may be restriction enzyme digested or directlysequenced for detection of said mutations.

In a second aspect, the invention provides a kit for diagnosis ofautosomal dominantly inherited isolated GH deficiency (IGHD II) in apatient sample, comprising means for analysing the presence of absenceof the missense mutation is G⁶¹⁹¹ to T in exon 4 of GH-1 which changesvaline 110 to phenylalanine.

The means may comprise reagents, primers etc for analysing a part of theGH-1 gene which includes this single nucleotide polymorphism.

Preferably, the kit also comprises means for analysing the presence orabsence of GH-1 splice site mutations causing skipping of exon 3 ofGH-1.

The splice site mutation is preferably a +2T to C transition of thesecond base of the intron 3 donor splice site.

The kit of the invention may comprise one or more specific GH-1 primerpairs selected from GH3.2 (nt 6578-6600), GH5.1 (nt 5503-5525); GH5.2(nt 5555-5577), GH3.4 (nt 6547-6568); GH5.7 (nt 581-5835), GH3.7 (nt6121-6140) and the following sequencing primers GS5.8 (nt 5629-5648),GS3.8 (6495-6515).

Alternatively to the sequencing primers, the kit may comprise one ormore of the following restriction enzymes MvnII, NlaIII, DdeI, MaeII.

Autosomal dominantly inherited isolated GH deficiency (IGHD II) iscaused by mutations of GH-1 which alter the normal structure of GH. Westudied 16 familial cases and one sporadic case with IGHD II from oneDutch and 4 German families by direct sequencing of PCR amplified gDNAand ectopic transcript analysis of lymphocyte mRNA. In addition, theclinical data of the affected individuals were analyzed. Two previouslyreported mutations and one novel splice site mutation in intron III ofGH-1 (+1G to C, +1G to A and new: +2T to C) were detected which causeexon 3 skipping. We also discovered a novel G⁶¹⁹¹ to T missense mutationin exon 4 of GH-1 which changes valine 110 which is highly conserved inmammalian and several non-mammalian GH to phenylalanine. Splicing of theprimary RNA transcript was not affected by this mutation which is verylikely to alter the normal GH structure at the protein level.

The onset of growth failure was earlier and the degree more severe inthe affected children with GH-1 splice site mutations in comparison tothose with the GH-1 missense mutation. In addition, the severity of thephenotype was inter-individually very variable, even within the samefamily. The age at diagnosis was between 0.8 and 9.6 years (median 5.1),height at diagnosis was between −2.5 and −8.1 SDscore (median −4.0).Most of the children were lean at diagnosis with a BMI ranging from −1.7to +3.3 SDscore (median −0.4). The 5 affected adults had final heightsbetween −1.8 and −4.5 SDscore (median −3.6), centripetal obesity andmuscular hypotrophy. Before therapy, IGF-I and IGFBP3 serum levels ofall affected children were severely diminished (median IGF I 15.1 μg/L,median IGFBP3 910 μg/L). The maximum GH peak in a total of 25stimulation tests was between 0. 1 and 5.0 μg/L (median 0.9) indicatingsevere GH deficiency. The height of the adenohypophysis studied by MRIwas normal in 2 affected children and mildly decreased in 2 others.Substitution with GH resulted in good catch-up growth in all treatedchildren. The discrepancy between the very homogeneous hormone dataproving severe GH- and IGF-I deficiency and the high variability ofgrowth failure, even within the same family, suggests that the onset andthe predominance of growth hormone dependent growth during infancy isindividually different. Children with severe GH- and IGFI deficiency,but normal size of the adenohypophysis should be examined for GH-1splice site and missense mutations.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described more closely below in association withthe accompanying drawings, in which:

FIG. 1 shows pedigrees of the previously reported Families 4 and 5.Filled symbols indicate the affected individuals.

FIG. 2 shows pedigrees of Families 1 and 2 and electrophoretic analysisof the amplified GH-1 fragment from family members (1-9) and normalcontrols (C) after restriction enzyme digestion with MvnI (Family 2) orNlalI (Family 1) whose recognition sites are underlined below. The bandof the undigested fragment is indicated by the arrow. Both heterozygotesplice site mutations generate one new recognition site for therespective enzyme which is evident by the appearance of two smallerbands after digestion. M =mol wt marker.

FIG. 3 shows a pedigree of Family 3 and electrophoretic analysis of theamplified GH-1 fragment from family members (1-9) and one normal control(C) after restriction enzyme digestion with MaeII whose recognition siteis underlined below. The band of the undigested fragment is indicated bythe arrow. Because the heterozygote mutation destroys the recognitionsite for MaeII, the DNA fragment of the affected family members isincompletely digested. M=mol wt marker.

FIG. 4 shows electrophoretic analysis of the amplified GH-1 cDNA. Lane 1contains the amplified cDNA from a patient with the GH-1 intron 3+1 G toC donor splice site mutation (Family 5) which shows in comparison to thenormal control (C) a shortening of the major fragment (arrow b)demonstrating loss of exon 3. The same analysis from one patient withthe novel G⁶¹⁹¹ to T missense mutation (V110F) is shown in Lane 2. Themain fragment is identical in size to the control's fragment (C)excluding missplicing (arrow a). M=mol wt marker.

FIG. 5 shows alignment of the human GH sequence (amino acids 104-117)with the sequences from mammalian and non-mammalian GHs. The valine atposition 110 is highly conserved and is located next to the N-terminalbeginning of the third α-helix. Human (SEQ ID NO: 1), rat (SEQ ID NO:2), ovine (SEQ ID NO: 3), porcine (SEQ ID NO: 4), bovine (SEQ ID NO: 5),chicken (SEQ ID NO: 6), and bullfrog (SEQ ID NO: 7).

FIG. 6 is a comparison of age and height SDscore at diagnosis of the 12children with IGHD II. The bars showing data from siblings have the samefilling. The black bars show the data from the family with V 110F GH.The high variability of growth failure in IGHD II, even within the samefamily, is clearly demonstrated.

DETAILED DESCRIPTION

The pedigrees of the five families (No. 1 to 5) are shown in FIGS. 1, 2and 3. Family 4 and Family 5 have been reported previously (5,17). Allaffected children were prepubertal at diagnosis and had normal f T4,TSH, prolactin and cortisol levels. Basal serum levels of IGF-1 andIGFBP3 were determined in all but one patient. Two independent GHstimulation tests were performed in 7 patients, only the insulin test in3 patients and no test because of young age (<1.0 year) in 2 patients ofwhich one had a pathologically low GH level during spontaneoushypoglycemia. Out of the 12 affected children, 11 were treated with rhGHsc (median dose 0.17 mg/kg per week). MR imaging with narrow scanning ofthe pituitary region and gadolinium injection was performed in 4patients and one affected parent from 3 families. Blood samples forgenetic analysis were taken after obtaining informed consent fromparents and patients.

Materials and Methods

Hormone Measurements

GH serum levels were measured in different clinical centers by severalassays (RIA, ELISA, EIA) which had the same cut-off level of 10 μg GH/Lfor the normal response to stimulation. IGF-I and IGFBP3 concentrationsin Families 1, 2, 3 and 5 were determined using the same assays by Blumet al. (18).

DNA and RNA Extraction

Genomic DNA was extracted from 5 ml frozen EDTA blood using anextraction kit (Genomix Blood Scale-Up, Talent, Triest, Italy) which wasbased on chloroform extraction after initial blood lysis. Total RNA fromperipheral lymphocytes was extracted according to the method describedby Chomczynski and Sacchi (19).

Oligonucleotide Primers

For the reverse transcription reaction, the used primer corresponded tonucleotides 6578-6600 (GH3.2) of the reported GH-1 sequence (20). ThePCR was performed using GH3.2 and the upstream primer 5503-5525 (GH5.1).The up-stream primer of the nested PCR corresponded to the nucleotides5555-5577 (GH5.2) and the down-stream primer to 6547-6568 (GH3.4). Forrestriction digest analysis with MvnII, NlaIII and DdeI, the nested PCRwas performed with the upstream primer GH5.7 (5816-5835) and thedownstream-primer GH3.7 (6121-6140). The used sequencing primers wereGS5.8 (5629-5648) and GS3.8 (6495-6515).

RT-PCR of RNA

RNA (5 μg) was reverse transcribed in PCR buffer and the total cDNA wasamplified by nested PCR as previously described (5). The PCR product (10μl of the reaction volume) were electrophoresed on 8% PAGE.

PCR of Genomic DNA

Genomic DNA was amplified by nested PCR. The first PCR was performedwith 0.2 μg gDNA, 100 pmol of each primer GH5. 1 and GH3, 2.5 U of TaqDNA polymerase (Qiagen, Hamburg, Germany), 0.2 mmol/1 of each dNTP inQiagen PCR buffer with a final volume of 50 μl. The reaction mixture wascycled 30 times (95° C., 60 s; 65° C., 60 s; 72° C., 90 s). An aliquotof this reaction (0.2 μl) was amplified in the nested PCR using theup-stream primer GH5.2 and the down-stream primer GH3.4 or alternatively(for restriction analysis) the sense-primer GH5.7 and the anti-senseprimer GH3.7.

Restriction Digest

The 325 bp fragment of GH-1 (5816-6140) containing the complete intron 3was digested with 20 U MvnII or with 20 U of NlaIII or with 10 U of DdeI(Boehringer Mannheim, Germany) in a volume of 30 μl containing 10 μI PCRproduct for 3 hrs. The 1014 bp fragment of GH-1 (5555-6568) containingthe genomic sequence from exon 2 to exon 5 was digested with 4 U MaeII(Boehringer Mannheim, Germany) under the same conditions. The fragmentswere visualized by ethidium bromide staining after run on an 8% PAGE.

Direct Sequencing

PCR products were directly double-stranded sequenced with the ThermoSequenase cycle sequencing kit containing 7-deaza-dGTP. The reaction wasperformed according to the manufacture's recommendations (Amersham,Germany). The sequencing primers were 5′-labelled with IR-800fluorescent dye. The products were run under denaturing conditions on aLi-COR DNA automatic sequencer 4200.

Results

Direct sequencing of the PCR-amplified genomic DNA resulted in thedetection of a heterozygote point mutation of the first base of thedonor splice site of intron 3 in the affected individuals of Families 1,4 and 5. In detail, we found a G to A transition in Family 1 and the Gto C transversion reported previously by us in Families 4 and 5 (5,17).A new splice site mutation with a T to C transition of the second baseof the intron 3 donor splice site was detected in Family 2. Restrictionfragment analysis of a 327 bp DNA fragment (5816-6140) containing thecomplete intron 3 with NlaIII (Family 1) and MvnI (Family 2) (FIG. 2),and with DdeI (Families 4 and 5) (5,17) detected the mutation in allaffected, but in no unaffected individuals of the 4 families. Ectopictranscript analysis of lymphocyte mRNA was performed in the proband ofFamily 5 and revealed the presence of a shortened mRNA lacking exon 3(FIG. 4, lane 1)(5).

In Family 3, we detected a novel heterozygous point mutation in exon 4with a G to T transversion at position 6191. Co-segregation with GHdeficiency were proven by restriction fragment analysis with MaeIIdemonstrating heterozygote loss of the MaelI recognition site in allaffected individuals (FIG. 3). Because the G⁶¹⁹¹ to T mutation wasinside an exonic motive that was homologous to a sequence which wasreported to act as an exonic splicing enhancer in the human IgM gene(21), we performed ectopic transcript analysis of peripheral lymphocyteRNA. The main GH mRNA fragment found was identical to controls excludinga defect of primary RNA splicing (FIG. 4, lane 2). The missense mutationresults in an amino acid exchange from valine to phenylalanine atposition 110 of the mutant growth hormone (V110F GH). The alignment ofGH sequences revealed that Val 110 is highly conserved in mammalian andalso in several non-mammalian GH (FIG. 5).

The clinical characteristics of the affected children at the time ofdiagnosis are summarized in Table 1. TABLE 1 Clinical characteristics of12 children with IGHD II at diagnosis. Quantitative values are given asmedian and range. Families 1, 2, 4, 5 Family 3 Sex (m/f) 4/2 4/2 Age(yrs) 2.3 (0.8 to 7.1) 6.3 (3.5 to 9.6) Height (SDscore) −4.7 (−2.5 to−8.1) −4.2 (−3.4 to −5.3) Height (cm/yr) 4.7 (3.8 to 6.0) 3.9 (2.5 to5.0) velocity BMI (SDscore) −0.5 (−1.5 to 3.3) −0.3 (−1.7 to 0.8) Boneage (yrs) 0.8 (0.2 to 5.5) 4.5 (1.5 to 8.0)

The SDscores were taken from Prader et al. (22). For better comparisonof the genotype-phenotype-relation, the clinical data from the childrenwith GH-1 splice site mutations (Families 1, 2, 4 and 5) and from thechildren affected by the GH-1 missense mutation (Family 3) are shownseparately. The age at diagnosis and the degree of short stature werevery variable in members of the same family as illustrated in FIG. 6.Overall, children with GH-1 splice site mutations showed a tendency toan earlier onset and more severe degree of growth failure in comparisonto the children with the GH-1 missense mutation (Table 1 and FIG. 6).Only two of the 12 affected children (5.1 and 7.1 years old) wereoverweight with a BMI SDscore of 2.2 and 3.3. The bone age was retardedin median by 1.6 years (range 0.5 to 3.6). The IGF-I and IGFBP3 levelswere pathologically low in all affected children (Table 2). TABLE 2Serum levels of the GH dependent factors at diagnosis. Values are givenas median and range. Families Family Age (yrs) IGF-1 (μg/L) IGFBP3(μg/L) 1, 2, 4, 5 (n=) 3 (n=) 0.8-2.5 8 (7-26) 340 (280-400) 3 0 3.5-6.014 (10-20) 860 (740-910) 1 3 6.5-9.6 27 (9-54)  1150 (990-1410) 1 329.5-58.5 48 (31-96)  775 (540-2670) 4 1

The stimulated GH levels were very low in all tested children regardlessof the stimulus (Table 3). A GH level above 3 μg/L was reached in 3 of25 stimulation tests (Table 3). TABLE 3 Growth hormone peak levels instimulation tests. Values are given as median and range. FamiliesStimulus Peak level (μg/L) 1, 2, 4, 5 (n=) Family 3 (n=) Insulin 1.1(0.2-5.0) 5 5 Arginine 0.6 (0.1-2.1) 6 0 Clonidine 1.7 (0.1-4.3) 2 2GHRH 2.1 (0.9-2.3) 3 0 Glucagon 0.8 (0.5-1.0) 2 0

Therapy with 0. 17 mg/kg rhGH sc weekly has been performed in 10children for more than two years. The median height velocity during thefirst 2 years of therapy increased to 10, 0 cm/year (range 7.0 to 12.5)which was equivalent of a median amplification of the growth velocity by2.1 fold (range 1.7 to 3.6). MR imaging of the hypophysis showed normalmorphology and normal adenohypophysal height according to Argyropoulouet al. (23) in 2 affected children from Families 2 and 5 (−0.5 and −0.6SDS) and a normal hypophysis with mildly reduced height in 2 childrenfrom Families 1 and 2 (−2.5 and −2.6 SDS) and in one adult (Family 1).The 5 affected adults with GH-1 splice site mutations (4 females) hadshort stature −3.6 (−2.7 to −4.2), centripetal obesity, muscularhypotrophy, but normal fertility. The IGF-I serum levels were severelyreduced (Table 2). The mother with V110F GH had short normal stature(−1.8 SDS), but also centripetal obesity and muscular hypotrophy. Herseven pregnancies were reportedly uneventful.

Discussion

All affected individuals of the five families whose pedigrees suggestedan autosomal dominant transmission of IGHD carried a deleterious pointmutation of GH-1 suggesting a very high prevalence of these mutations inIGHD II. One novel splice site mutation found in one family affected thehighly conserved second base of the intron 3 donor splice site (+2T toC). The predicted effect of this mutation as it was shown for mutationsof the first base of intron 3 is the skipping of exon 3 during splicing(4,5). The resultant del32-71 GH exerts a dominant negative effect onthe wild-type GH which is cell-specific and not observed innon-secretory cell types like EBV-transformed lymphocytes (7) orCOS-cells (8,9). Heterodimer formation between wild-type GH and del32-71GH which lacks one cysteine at position 53 was the first hypotheticalproposal for the dominant negative effect (4). This theory has recentlybeen questioned by the finding that the recombinant double mutantdel32-71,C165A GH which lacks the unpaired cysteine at position 165 hadthe same effect in-vitro (9). Both, del32-71 GH and wild-type GH do notaccumulate in neuroendocrine cell lines indicating a decrease inintracellular stability (9) whose molecular basis is still unknown (24).In this context, it is of importance that missense mutations of the GH-1also result in a mutant GH with a dominant negative effect.

The present inventors report for the first time the V110F mutation ofGH-1. Its genetic basis is a C to T transition in a CpG dinucleotidewhich is a general hotspot for mutations in vertebrate genomes (25).This mutation changes a valine which is completely conserved inmammalians, and in some amphibians and birds, to phenylalanine. Valineis located next to the N-terminal beginning of the third alpha-helix andintegrated in the closely packed core of the four-α-helix-bundle of GH(26). The more bulky phenylalanine at this position is very likely tointerfere with the normal folding of GH. Two other GH1 missensemutations which co-segregate with IGHD II were recently reported: P89Land R183H (15, 16). Similar to V110F, both mutations change highlyconserved amino acids. Proline 89 forms a kink in the second a-helix ofGH which leucine is not able to form (24). Arginine 183 is next to thedisulfide bond of cysteine 182 whose formation may be disturbed by themore bulky histidine (24). However, the effect of the two missensemutations in cultured neuro-endocrine cells has not yet been shown.

In the past, screening for GH-1 defects was performed if severe growthfailure with a height below −4.5 SDS at diagnosis were present (6).Severe short stature according to this definition was only present inone third of our affected individuals at diagnosis indicating thatgrowth failure in IGHD II is less severe than would be expected. Thechildren with the splice site mutations were younger and shorter atdiagnosis than their counterparts with the missense mutation. Moderategrowth failure was also reported from the family with the P89L mutation(16). In addition, we observed in the three families in which more thanone child was affected a pronounced intrafamilial variability betweensiblings in both mutation groups with height differences exceeding 3.0SDscore. This variability in growth did not correlate with the severityof growth hormone or IGF-I deficiency because the hormone levels werevery low in all tested subjects including those affected individualswith the highest growth velocity and only minor growth failure. There isno substantial evidence for a course of slow progression of GHdeficiency in IGHD II like it was described for the dominantly inheritedvasopressin deficiency (24). Other factors than systemic GH- andIGF-I-levels must be responsible for the differences in growth failure.These unknown factors obviously modulate the start and predominance ofGH dependent growth. Some of these unknown factors may also be involvedin the high variability of growth failure reported in Laron syndrome(27).

Data on the systematic examination of the pituitary anatomy inmonogenetic disorders are scarce (29). Approximately 50% of thehormone-producing cells of the anterior pituitary are somatotropic cells(29). Recent MRI observations in children with idiopathic GHD suggesteda positive relationship of the volume of the adenohypophysis and thesecretory GH capacity (30). This is not the case in IGHD II: the 4children examined by MRI showed a normal adenohypophysis in 2 cases,mild hypoplasia in the two others. Normal size of the anteriorhypophysis was also reported in two children affected with IGHD IAsuggesting that the presence of GH is not a pre-requisite for normalsize of the adenohypophysis (31). Therefore, the combination of normalor almost normal height of the adenohypophysis in the presence of severeGH and IGF-1 deficiency is highly suggestive of the presence of acausative GH-1 mutation. In such cases, the molecular diagnosisestablishes the basis for genetic counselling and for the recommendationof a live-long substitution with GH.

REFERENCES

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1. A kit for diagnosis of autosomal dominantly inherited isolated GHdeficiency (IGHD II) in a patient sample, comprising means for analyzinga patient sample for the presence or absence of a T at nucleotideposition 6191 in exon 4 of the GH-1 gene.
 2. A kit according to claim 1,also comprising analyzing the presence or absence of a GH-1 splice sitemutation causing skipping of exon 3 of the GH-1 gene.
 3. A kit accordingto claim 2, wherein the means for analyzing are for analyzing a splicesite mutation which is a +2T to C transition of the second base of theintron 3 donor splice site.
 4. A kit according to claim 1, comprisingone or more specific GH-1 primer pairs selected from the groupconsisting of GH3.2 (nt 6578-6600 of the GH-1 gene), GH5.1 (nt 5503-5525of the GH-1 gene); GH5.2 (nt 5555-5577 of the GH-1 gene), GH3.4 (nt6547-6568 of the GH-1 gene); and GH5.7 (nt 5816-5835 of the GH-1 gene),GH3.7 (nt 6121-6140 of the GH-1 gene).
 5. A kit according to claim 2,comprising one or more specific GH-1 primer pairs selected from thegroup consisting of GH3.2 (nt 6578-6600 of the GH-1 gene), GH5.1 (nt5503-5525 of the GH-1 gene); GH5.2 (nt 5555-5577 of the GH-1 gene),GH3.4 (nt 6547-6568 of the GH-1 gene); and GH5.7 (nt 5816-5835 of theGH-1 gene), GH3.7 (nt 6121-6140 of the GH-1 gene).
 6. A kit according toclaim 3, comprising one or more specific GH-1 primer pairs selected fromthe group consisting of GH3.2 (nt 6578-6600 of the GH-1 gene), GH5.1 (nt5503-5525 of the GH-1 gene); GH5.2 (nt 5555-5577 of the GH-1 gene),GH3.4 (nt 6547-6568 of the GH-1 gene); and GH5.7 (nt 5816-5835 of theGH-1 gene), GH3.7 (nt 6121-6140 of the GH-1 gene).
 7. A kit according toclaim 1, comprising sequencing primers GS5.8 (nt 5629-5648 of the GH-1gene), GS3.8 (nt 6495-6515 of the GH-1 gene).
 8. A kit according toclaim 2, comprising sequencing primers GS5.8 (nt 5629-5648 of the GH-1gene), GS3.8 (nt 6495-6515 of the GH-1 gene).
 9. A kit according toclaim 3, comprising sequencing primers GS5.8 (nt 5629-5648 of the GH-1gene), GS3.8 (nt 6495-6515 of the GH-1 gene).
 10. A kit according toclaim 4, comprising sequencing primers GS5.8 (nt 5629-5648 of the GH-1gene), GS3.8 (nt 6495-6515 of the GH-1 gene).
 11. A kit according toclaim 1, comprising one or more of the restriction enzymes MvnII,NlaIII, DdeI, and MaeII.
 12. A kit according to claim 2, comprising oneor more of the restriction enzymes MvnII, NlaIII, DdeI, and MaeII.
 13. Akit according to claim 3, comprising one or more of the restrictionenzymes MvnII, NlaIII, DdeI, and MaeII.
 14. A kit according to claim 4,comprising one or more of the restriction enzymes MvnII, NlaIII, DdeI,and MaeII.